Practical Report: Determination of Concentration Using a Standard Curve

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Practical report: Determination of concentration using a standard curve

Introduction Spectrophotometer is an instrument that confirms the measurement of specified wavelength of light that passes through medium. The measurement of light absorbance by a solution is also done with spectrophometer. Absorption spectrum Spectrometers are mainly and widely functional in identifying the components of solutions that helps determine the components’ concentrations. Compounds are experimentally identified differently through absorption characters. The absorbance of a solution at a certain wavelengths pertains to concentration of absorbing materials in the cell. The Lambert law explains and describes that the concentration of a solution can be determined by considering the relationship of absorbance and concentration. Aims or Objectives To have knowledge, experience and be most familiar with the use of spectrophometer To have knowledge and experience of tubes and other relevant laboratory facilities needed. 1

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To learn to independently prepare, conduct, and record laboratory practicals or demonstrations of many solutions of known concentration. To accurately measure the absorbance at its correct maximal wavelength at which it absorbs. To learn to plot a standard curve of absorbance and concentration To learn to determine the unknown solution concentration by considering or through standard curve Chemical Agents, Materials, and Methods The chemical agents written to procedurally follow were 1. 0.004 mM standard phenolphthalein 2. Unknown phenolphthalein solution 3. 0.12 M glycine-phosphate buffer, PH 11.2 I began this experiment by carefully ejecting eight tubes from their cupboard storages. I marked and arranged the tubes from one to eight. Next step was to fill up a beaker with de-ionized (distilled) water. In order to complete a protocol (table) in the prac booklet, I carefully used pipettes to add de-ionized water to tubes using the required measurements below;

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Tube1 (2.5 ml), Tube2 (2.0 ml), Tube3 (1.5 ml), Tube4 (1.0 ml), Tube5 (0.5 ml), Tube6 (0 ml), Tube7 (0 ml), Tube8 (0 ml) I used a new pipette to add 0.04 mM standard phenolphthalein solution using the required measurements below, Tube1 (0 mM), Tube2 (0.5 mM), Tube3 (1.0 mM), Tube4 (1.5 mM), Tube5 (2.0 mM), Tube6 (2.5 mM), Tube7 (NA), Tube8 (NA) I again used a new pipette to add the “Unknown” Phenolphthalein solution (ml) using the required measurements below; Tube1 (NA), Tube2 (NA), Tube3 (NA), Tube4 (NA), Tube5 (NA), Tube6 (NA), Tube7 (2.5 ml), and Tube8 (2.5 ml) The last pipette for the practical was used to add glycine buffer. We added 5 ml of glycine buffer into each of the eight tubes. I lastly confirmed the measurement of light of absorbance by a solution in each of the eight tubes by using spectrophometer. Results After supplying the tubes with all reagents, the protocol is completed in the table below; Solution Tube

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1 Water (ml) 0.004 mM standard phenolphthalein “Unknown” Phenolphthalein solution (ml) Total Volume per tube of glycine Buffer (ml) Final conc. Of phenolphthalein in the tube (mM) Absorbance (550 nm) 0.00 5 NA 2.5 0

2 2.0 0.5

3 1.5 1.0

4 1.0 1.5

5 0.5 2.0

6 0 2.5

7 0 NA

8 0 NA

NA

NA

NA

NA

NA

2.5

2.5

5

5

5

5

5

5

5

0.014 0.0008 0.0016 0.0024 0.0032 0.004

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?

0.200

0.430

0.630

0.830

0.998

0.65

0.65

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Conclusion As you can see above, the protocol (table) is accurately and correctly completed. In the final concentration of phenolphthalein in the tube, I have used 0.0008 because we are using 0.004 mm. Some of the tubes were supplied zero reagents to cover background level. The concentration of the unknown could be out of the range of standards of its 550nm if the practical was not performed well. The concentration of the phenolphthalein is adjusted to PH 11 to avoid straight pink colour.

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